Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Equivalent Aqueous Phase Modulation of Domain Segregation in Myelin Monolayers and Bilayer Vesicles

Purified myelin can be spread as monomolecular films at the air/aqueous interface. These films were visualized by fluorescence and Brewster angle microscopy, showing phase coexistence at low and medium surface pressures (<20-30 mN/m). Beyond this threshold, the film becomes homogeneous or not, depending on the aqueous subphase composition. Pure water as well as sucrose, glycerol, dimethylsulfoxide, and dimethylformamide solutions (20% in water) produced monolayers that become homogeneous at high surface pressures; on the other hand, the presence of salts (NaCl, CaCl₂) in Ringer's and physiological solution leads to phase domain microheterogeneity over the whole compression isotherm. These results show that surface heterogeneity is favored by the ionic milieu. The modulation of the phase-mixing behavior in monolayers is paralleled by the behavior of multilamellar vesicles as determined by small-angle and wide-angle x-ray scattering. The correspondence of the behavior of monolayers and multilayers is achieved only at high surface pressures near the equilibrium adsorption surface pressure; at lower surface pressures, the correspondence breaks down. The equilibrium surface tension on all subphases corresponds to that of the air/alkane interface (27 mN/m), independently on the surface tension of the clean subphase.     

Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten

Hydrolysate of extruded corn gluten with higher solubility and antioxidative property was prepared. Extrusion and starch removal of corn gluten were applied as pretreatment before enzymatic hydrolysis by Alcalase. The amylase hydrolysis of starch at 70°C for 3 h resulted in the removal of the starch from the extruded corn gluten. The best hydrolysis results can be obtained by conducting the hydrolysis at 60°C with water addition 20 g/g protein, enzyme addition 0.048 Ansen units/g protein, pH 8.5, and 120 min. Degree of hydrolysis of extruded and nonextruded corn gluten reached 39.54 and 31.16%, respectively, under the optimal condition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the optimal hydrolysate revealed that proteolysis of extruded corn gluten was more extensive than proteolysis of its counterpart which was not subjected to extrusion. The molecular weight of the peptides in the optimal hydrolysate was mainly over 3,710–660 Da as determined by gel filtration chromatography. The hydrolysates displayed good solubility and antioxidative activity. The separation profile of the hydrolysate on an ion exchange chromatography of Q-Sepharose Fast Flow showed that many kinds of peptides had antioxidative effect. A new peptide with antioxidative activity was purified, and its amino acid sequence was Phe-Pro-Leu-Glu-Met-Met-Pro-Phe, which was identified by Q-TOF2 mass spectrometry.     

Monitoring of enzymatic hydrolysis of starch by microdialysis sampling coupled on-line to anion exchange chromatography and integrated pulsed electrochemical detection using post-column switching

A quantitative evaluation of the hydrolysis of wheat starch using Termamyl, a thermostable alpha-amylase (endo-1,4-alpha-d-glucan, glucanohydrolase; EC 3.2.1.78), is reported. Data from the monitoring of the hydrolysis of wheat starch indicated that, after 1 h, glucose and maltooligosaccharides up to DP 7 were the main hydrolysis products and thus enabled optimization of a liquefication step during the production of L-lactic acid. The monitoring system used, both in the on- and off-line mode, was based on continuous flow microdialysis sampling (CFMS) coupled to anion exchange chromatography and integrated pulsed electrochemical detection (IPED). A microdialysis probe equipped with a 5-mm polysulfone (SPS 4005) membrane, with a molecular-weight cut-off of 5 kDa, was used to sample the hydrolysis products of native wheat starch at 90 degrees C. Characteristic fingerprint separations were achieved by anion exchange chromatography after enzymatic hydrolysis. Post-column switching improved the detection and, consequently, also quantification of the hydrolysates as fouling of the electrode could be reduced. Maltooligosaccharide standards were used for quantification and to verify the elution of the hydrolysates by spiking the off-line samples. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 546-554, 1997.     

Influence of stearic acid monolayers upon the procaine adsorption from underlying alkaline aqueous solutions

Adsorption of procaine at the air/water interface and its penetration into stearic acid monolayers from aqueous subphase of pH 8 are studied by measuring surface tension of aqueous procaine solutions and by recording surface pressure vs. mean molecular area curves for stearic acid monolayers spread onto procaine solutions of different concentrations. The amount of procaine in the interface is derived by means of Gibbs' equation. Results are compared to those obtained earlier at pH 2 and on unbuffered subphases. With increasing pH an increasing procaine adsorption and procaine penetration is observed. This phenomenon is interpreted in terms of protolytic equilibria in which participate both surfactants procaine and stearic acid.     

The Equilibrium of Phosphatidylcholine–Amino Acid System in Monolayer at the Air/water Interface

Monolayers of phosphatidylcholine, tyrosine, and phenylalanine and binary mixtures phosphatidylcholine–tyrosine or phosphatidylcholine–phenylalanine were investigated at the air/water interface. Phosphatidylcholine (lecithin, PC), tyrosine (Tyr), and phenylalanine (Phe) were used in the experiment. The surface tension values of pure and mixed monolayers were used to calculate π–A isotherms. The surface tension measurements were carried out at 22°C using an improved Teflon trough and a Nima 9000 tensiometer. The Teflon trough was filled with a subphase of triple-distilled water. Known amounts of lipid dissolved in 1-chloropropane were placed at the surface using a syringe. The interactions between lecithin and amino acid result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants of PC–Tyr as well as PC–Phe complexes. We considered the equilibrium between the individual components and the complex and established that lecithin and amino acid formed highly stable 1:1 complex.     

Protein adsorption at the oil/water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements.

The dynamics of protein adsorption at an oil/water interface are examined over time scales ranging from seconds to several hours. The pendant drop technique is used to determine the dynamic interfacial tension of several proteins at the heptane/aqueous buffer interface. The kinetics of adsorption of these proteins are interpreted from tension/log time plots, which often display three distinct regimes. (I) Diffusion and protein interfacial affinity determine the duration of an initial induction period of minimal tension reduction. A comparison of surface pressure profiles at the oil/water and air/water interface reveals the role of interfacial conformational changes in the early stages of adsorption. (II) Continued rearrangement defines the second regime, where the resulting number of interfacial contacts per protein molecule causes a steep tension decline. (III) The final regime occurs upon monolayer coverage, and is attributed to continued relaxation of the adsorbed layer and possible build-up of multilayers. Denaturation of proteins by urea in the bulk phase is shown to affect early regimes.     

Monolayers of long chain lecithins at the air/water interface and their hydrolysis by phospholipase A2

The behavior of phosphatidylcholine monolayers at the air/water interface was studied by measuring their surface isotherm, surface potential, surface viscosity, and rate of hydrolysis by the dimeric phospholipase A2 from the venom of Crotalus atrox. The monolayers showed typical liquid-expanded behavior. In this phase, the surface potential was linearly dependent on surface concentration and extrapolated at zero concentration to a value characteristic of a liquid hydrocarbon/water interface. The rate of the reaction was measured by monitoring changes in area at constant surface pressure for 1,2-dioctanoyl- and 1,2-didecanoyl-3-sn-phosphatidylcholines, and by monitoring changes in surface potential for 1,2-dimyristoyl-, 1,2-dipalmitoyl-, 1-palmitoyl-2-oleoyl-, and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholines. The enzymatic hydrolysis is first order with respect to the enzyme-calcium complex which forms with a Kd = 1.5 mM. A mechanism is proposed to account for the dependency of the reaction rates on the surface concentration of the substrate. We postulate that the rate-limiting step is the decomposition of a quaternary complex formed from two phospholipid molecules, one calcium ion and one dimeric enzyme. The rate is independent of the surface pressure per se; addition of inert lipids to a monolayer at constant area, and hence constant surface concentration of the substrate, increases the surface pressure without changing the surface density of the substrate yielding maximal enzymatic rate. The enzyme is specific for loosely packed substrate molecules in the liquid-expanded state: transition into the liquid-condensed state or compression of the liquid-expanded layer beyond 80 A2/phospholipid strongly inhibits the enzymatic reaction. Our results show that surface recognition is a direct consequence of a bifunctional active site since it is only at a phospholipid surface that the distance between two substrate molecules is optimal for forming a catalytically competent enzyme-Ca2+-(substrate)2 complex.     

Supramolecular assembly using helical peptides

We investigated supramolecular assemblies of various hydrophobic helical peptides. The assemblies were formed at the air/water interface or in aqueous medium. The hexadecapeptide, Boc-(Ala-Aib)₈-OMe (BA16M), was reported to take α-helical structure by X-ray analysis. Several derivatives were prepared, which have the repeating sequence of Ala-Aib, Lys(Z)-Aib or Leu-Aib, or have the terminal chemically modified. CD spectra of the peptides indicated helical conformation in ethanol solution. The surface pressure-area isotherms of the peptide monolayers showed an inflection at the surface area corresponding to the cross section along the helix axis, and the monolayers were collapsed by further compression. All the helical peptides oriented their helix axis parallel to the air/water interface on the basis of the results of transmission IR spectra and RAS of the monolayers transferred onto substrates.A small mound was observed in the isotherm of BA16M and other derivatives, which was ascribed to the phase transition from the liquid state to the solid state. One mol% of FITC-labeled peptide was mixed into the monolayers to visualize the phase separation of the solid and liquid states at the surface pressure of the coexisting region. Various shapes of the dark domain were observed at the top of the mound in the isotherms by fluorescence microscopy. The helical peptides formed two-dimensional crystals at the air/water interface when they were compressed to the solid state.An amino-terminated helical peptide, HA16B, was suspended in an aqueous medium by a sonication method and transparent dispersion was obtained. The dynamic light scattering measurement of the dispersion revealed the particle size of 75 nm with a narrow size distribution. The molecular assembly of the helical peptide in water was called “Peptosome”, because it takes a vesicular structure.     

Structural characteristics of hydrolysates of proteins from extracted sunflower flour at the air-water interface

The structural and topographical characteristics of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH = 5.62%, 23.5%, and 46.3%) spread at the air-water interface at pH 7 and 20 degrees C were determined from pi-A isotherms coupled with Brewster angle microscopy (BAM). The structural characteristics of SP hydrolysate spread monolayers depend on the degree of hydrolysis. We observed a significant shift of the pi-A(APPARENT) isotherms toward lower molecular areas as the degree of hydrolysis (DH) increased. This phenomenon was attributed to spreading of the protein at the interface, especially at DH 46.3%. A change in the monolayer structure was observed at a surface pressure of 12-15 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized near the monolayer collapse and during the monolayer expansion proved the existence of large regions of protein aggregates. Reflectivity increased with surface pressure and was a maximum at the monolayer collapse. The monolayer thickness decreased as the degree of hydrolysis increased. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis.     

Interfacial tension of phosphatidylcholine-cholesterol system in monolayers at the air/water interface

Interfacial tension of an egg lecithin-cholesterol system was measured across the whole concentration range. Surface pressure-area isotherm measurements were carried out in a Langmuir trough at the air/water interface at room temperature (22 degrees C). The interfacial tension of the air/water interface was divided into contributions of components. The interfacial tension of a 1:1 complex between phosphatidylcholine and cholesterol was calculated. Its value equals 18 mN/m. The difference between the stability constant of 1:1 complex in the bilayer and the monolayer at the air/water interface is discussed.     

Cereal-based biorefinery development: integrated enzyme production for cereal flour hydrolysis

Restructuring the traditional fermentation industry into viable biorefineries for the production of fuels, chemicals and plastics is essential in order to replace (petro)chemical processing. This work presents engineering aspects of Aspergillus awamori submerged fermentation for on-site production of an enzymatic consortium that contains glucoamylase, protease and phosphatase. The crude broth filtrate was used for the production of wheat flour hydrolysates. Improvements on traditional starch hydrolysis carried out in two stages (liquefaction and saccharification) were attempted through integration of unit operations and reduction of processing temperature and reaction duration. An initial increase of temperature to 68 degrees C and a subsequent decrease to 60 degrees C for the rest of the enzymatic hydrolysis resulted in a starch to glucose conversion yield of 94 and 92% when a wheat flour concentration and commercial starch concentration of 225 g L(-1) was used, respectively. The use of crude broth filtrates resulted in the simultaneous hydrolysis of wheat protein and phytic acid, as was indicated by the increase in free amino nitrogen and phosphorus concentration, respectively.     

Reconstruction of lignin and hemicelluloses by aqueous ethanol anti‐solvents to improve the ionic liquid‐acid pretreatment performance of Arundo donax Linn

Ionic liquid (IL)‐acid pretreatment is known to not only enhance the enzymatic hydrolysis efficiency of lignocellulose but also to generate deposits on the surface of fiber by conventional water regeneration, which retard the increment. In this study, ethanol aqueous solution regeneration was developed as a new method to change the substrates characteristics for IL‐acid pretreatment and their effects on the enzymatic hydrolysis were evaluated. Following the IL‐acid reaction, the biomass slurry was subjected to ethanol aqueous solution at various concentration. Results indicated that anti‐solvent choice significantly influenced the reconstruction of both hemicelluloses and lignin as a result of the competition between water and ethanol. The partial removal of hemicelluloses and suitable lignin re‐localization contributed to a more porous structure. Consequently, the cellulose digestibility of aqueous ethanol regenerated samples was dramatically enhanced to ～100% and approximately 11‐ and 2‐fold higher than that of untreated and conventional water regenerated pretreated samples, respectively. A giant leap in the initial rate of enzymatic hydrolysis was also detected in 50% ethanol aqueous solution regenerated samples and only about 10 hr was needed to convert 80% of cellulose to glucose due to the appearance of cellulose II hydrate‐like and more porous structure.     

Enzymatic hydrolysis of chitosan films in water and physiological solution

It was shown that the processes of enzymatic hydrolysis of chitosan in aqueous acetic acid and on the surface of chitosan films in a solution of hyaluronidase in acetic acid are described by uniform kinetic constants. Kinetic parameters of enzymatic hydrolysis of the chitosan film samples in water and in physiological solution (Ringer–Locke’s solution) were determined. It was found that the introduction of medicinal agents and low-molecular-weight electrolytes to a chitosan-based film material reduces the rate of enzymatic hydrolysis of the films, which may indicate a possible increase in their service life when used on the wound surface.     

Application of infrared spectroscopy (attenuated total reflection) for monitoring enzymatic activity on substrate films

Infrared film analysis, a method based on infrared spectroscopy in the mode of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), is demonstrated as a novel analytical method for monitoring enzymatic activity on surface-attached substrate films in the mid infrared range (400-4000 cm(-1)). The ATR-FTIR technique is sensitive to molecules within a distance of approximately 1 microm from the ATR sampling unit surface (a 7 cm(2) hydrophobic ZnSe crystal). Applying a 0.2-0.3 microm thick film on the ATR unit surface, any chemical changes within this film as well as at the interface can be continuously monitored, even having an aqueous phase on top of the film. Infrared film analysis is considered especially useful for studying detergent enzymes, which act on surface bound films consisting of food component like vegetable oils (triacylglycerols) and carbohydrates (e.g. starch). Experimental data are presented for hydrolysis of a triacylglycerol film (triolein) by use of a triacylglycerol lipase (cutinase), and starch film degradation by use of an alpha-amylase.     

High consistency enzymatic hydrolysis of hardwood substrates

The feasibility of using a laboratory peg mixer to carry out high consistency enzymatic hydrolysis of lignocellulosic substrates was investigated. Two hardwood substrates, unbleached hardwood pulp (UBHW) and organosolv pretreated poplar (OPP), were used in this study. Hydrolysis of UBHW and OPP at 20% substrate consistency led to a high glucose concentration in the final hydrolysate. For example, a 48 h enzymatic hydrolysis of OPP resulted in a hydrolysate with 158 g/L of glucose. This is the highest glucose concentration ever obtained from enzymatic hydrolysis of lignocellulosic substrates. Fermentation of UBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations, 50.4 and 63.1 g/L, respectively after fermentation. Our results demonstrate that using common pulping equipment to carry out high consistency hydrolysis can overcome the rheological problems and greatly increase the sugar and ethanol concentrations after the hydrolysis and fermentation.     

How a fungus escapes the water to grow into the air

Fungi are well known to the casual observer for producing water-repelling aerial moulds and elaborate fruiting bodies such as mushrooms and polypores. Filamentous fungi colonize moist substrates (such as wood) and have to breach the water-air interface to grow into the air. Animals and plants breach this interface by mechanical force. Here, we show that a filamentous fungus such as Schizophyllum commune first has to reduce the water surface tension before its hyphae can escape the aqueous phase to form aerial structures such as aerial hyphae or fruiting bodies. The large drop in surface tension (from 72 to 24 mJ m-2) results from self-assembly of a secreted hydrophobin (SC3) into a stable amphipathic protein film at the water-air interface. Other, but not all, surface-active molecules (that is, other class I hydrophobins and streptofactin from Streptomyces tendae) can substitute for SC3 in the medium. This demonstrates that hydrophobins not only have a function at the hyphal surface but also at the medium-air interface, which explains why fungi secrete large amounts of hydrophobin into their aqueous surroundings.     

Hydroxynitrile lyase at the diisopropyl ether/water interface: evidence for interfacial enzyme activity.

A novel recycle reactor has been designed to determine the interfacial activity of hydroxynitrile lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity toward mandelonitrile cleavage is continuously measured in the reactor by following benzaldehyde product formation in the DIPE organic phase with an optical flow cell. For the first time, we establish that this enzymatic reaction is carried out by the hydroxynitrile lyase residing at the organic solvent/water interface and not in the aqueous bulk phase. Hydroxynitrile lyase adsorbs at the interface and exhibits extraordinary stability. Denaturation does not occur over several hours, although the surface pressure increases under the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzyme activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium constant of 0.032 L mg(-1). For the mandelonitrile-cleavage reaction at ambient temperature, p-Hnl follows Michaelis-Menten kinetics at the interface with a Michaelis constant of 14.4 mM and a specific activity close that for the bulk aqueous phase.     

Dynamic interfacial properties of human apolipoproteins A-IV and B-17 at the air/water and oil/water interface

Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/water interface apolipoproteins A-IV and B-17 displayed wide area - tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 3 10(-)(3) sec(-)(1), whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 3 10(-)(3) sec(-)(1). Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure.We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles.     

The Equilibria of Sphingolipid-Cholesterol and Sphingolipid–Sphingolipid in Monolayers at the Air–Water Interface

Monolayers of sphingomyelin (SM), ceramide (Cer) and cholesterol (Ch) and binary mixtures SM–Ch, SM–Cer and Cer–Ch were investigated at the air–water interface. SM, Cer and Ch were used in the experiment. The surface tension values of pure and mixed monolayers were used to calculate π-A isotherms. Surface tension measurements were carried out at 22 °C using a Teflon trough and a Nima 9000 tensiometer. Interactions between sphingolipid and Ch as well as sphingolipid and another sphingolipid result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air–water interface was developed in order to obtain the stability constants and Gibbs free energy values of SM–Ch, SM–Cer and Cer–Ch complexes. We considered the equilibrium between the individual components and the complex and established that sphingolipid and Ch as well as sphingolipid and another sphingolipid formed highly stable 1:1 complexes.